European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Cytogenetics
of FMR1 gene. Population screening is highly justifiable but no costeffective
technical approach has been developed. Normal individuals
show a range of allele sizes from 8 to 40 repeats with 20-22 and 29-
31 being the most common allele lengths. Accurate measurement of
allele size is crucial for diagnostic purposes and uses a combination
of Polymerase Chain Reaction (PCR) and Southern Blot (SB) techniques.
The latter is required to identify large alleles but it is expensive,
time-consuming and requires a large quantity of DNA. One particular
use of SB is distinction of normal homozygous alleles in females,
which are found in approximately 30% of cases, from pre-mutation
and full mutations. We developed a more sensitive PCR assay, which
distinguishes alleles differing by only one repeat. Reanalysis of female
DNA samples alleletyped as homozygous using a previous PCR assay
has shown that 50% are actually heterozygous and therefore do not
need to be verified using SB. This method has considerable advantages
compared with other diagnostic tests and greatly facilitates testing
of large numbers of samples.
P0340. Loss of methylation in imprinting control region
KVLQT1OT in first-trimester human spontaneous abortions
I. N. Lebedev, E. A. Sazhenova;
Institute of Medical Genetics, Tomsk, Russian Federation.
Genomic imprinting plays a significant role in regulation of early embryo
development. The search of imprinted genes mutations in human miscarriages
is a way to determine its effects. However, there are little evidences
about high level of imprinting alterations in spontaneous abortions,
except of hydatidiform mole and single instances of uniparental
disomy. We have suggested that expected negative effect of imprinting
disruption could be realized through epimutations of imprinted loci
rather than rare events of uniparental inheritance. The global changes
in genome methylation during preimplantation development as well as
a growing number of reports about association of assisted reproductive
technologies and imprinting disorders reinforce our suggestion.
In the present study we have performed methylation analysis of three
imprinting control regions (SNURF-SNRPN, H19, KVLQT1OT) and imprinted
gene CDKN1C in 84 first-trimester spontaneous abortions by
methyl-specific or methyl-sensitive PCR. Two tissues (extraembryonic
mesoderm and cytotrophoblast) with different patterns of epigenetic
reprogramming were investigated. Twenty four induced abortions were
studied as a control group. Differential DNA methylation of SNURF-
SNRPN, H19 and CDKN1C was registered in all cases. As to KVLQ-
T1OT, loss of methylation (LOM) in maternal allele was found in 8 miscarriages
(9.5%). Surprisingly, LOM was confined by extraembryonic
mesoderm or cytotrophoblast in 5 and 4 embryos, respectively. To our
knowledge this is a first report about epimutations of imprinting control
region in human miscarriages. Moreover, tissue-specific restriction of
LOM allows suggesting independent sporadic epigenetic aberrations
in different embryonic germ layers. This study was supported by RFBR
(N 05-04-48129) and FASI (N 02.512.11.2055).
P0341. CGH-array study of 40 holoprosencephaly patients
C. Bendavid 1 , C. Dubourg 1,2 , I. Gicquel 1 , J. Seguin 1 , L. Pasquier 3 , C. Henry 4 , S.
Odent 1,3 , V. David 1,2 ;
1 UMR6061 CNRS, Rennes, France, 2 Génétique Moléculaire, CHU, Rennes,
France, 3 Génétique Médicale, CHU, Rennes, France, 4 Cytogénétique, CHU,
Rennes, France.
Holoprosencephaly (HPE) is the most common developmental brain
anomaly in humans, usually associated with facial features. Our group
focuses on patients with HPE and normal karyotype. We previously
reported our results for point mutations screening and gene dosage in
HPE genes (SHH, SIX3, ZIC2 and TGIF), describing genomic defects
in up to 27% fetuses and 30% newborns. Recently, we screened subtelomeres
by MLPA and found alterations in known HPE candidate loci
but also in new regions, including many unbalanced translocations.
Therefore, we decided to screen HPE patients using CGHarray to assess
the frequency, location and size of such disorders.
We used Agilent CGH-array 44K technology. Out of 40 patients, we
screened 10 DNA samples with known quantitative anomalies and 30
samples with no alterations identified to date. First we localized the
breakpoints of the 10 DNA with known alterations (loss and/or gain in
specific loci) and showed no correlation between the size and severity
of the defect. Out of the 30 DNA with no genetic aetiology, we found
5 new rearrangements involving new potential HPE loci located on
110
chromosomes 21q, 20p, 15q, 6q, 5q, 17q and 18q. We observed both
isolated or associated genomic loss and gain, the latter suggesting
an unbalanced translocation from parental origin. Detected alterations
ranged from less than 1Mb to 16 Mb. Our data strongly reinforce the
multigenic and multihit origin in HPE and participate in the explanation
for the wide phenotypic spectrum described in this developmental
disorder.
P0342. High Resolution CGH (HR-CGH) in the diagnosis of
patients with mental retardation, dysmorphic features and
normal karyotype
S. Psoni 1 , G. Leventopoulos 1 , G. Bardi 2 , D. Iakovaki 2 , V. Papassava 2 , S.
Kitsiou-Tzeli 1 , A. Mavrou 1 , E. Kanavakis 1 , H. Fryssira 1 ;
1 Medical Genetics/Choremio Research Laboratory/Univ. of Athens/St Sophia’s
Children Hospital, Athens, Greece, 2 BIOANALYTIKI-GENOTYPE Research and
Applied Molecular Cytogenetics Center, Athens, Greece, Athens, Greece.
Introduction: Mental retardation (MR) is a common disorder, but etiologic
factors are not revealed in about half of the cases. CGH (Comparative
Genomic Hybridisation) techniques have been proved useful
not only in cancer genetics, but also in the diagnosis of MR . HR-CGH
is a whole-genome screening technique which detects cryptic subtelomeric
or interstitial chromosomal aberrations, not visible by conventional
cytogenetic studies.
Subjects-Methods: Five patients, (3 boys-2 girls, aged 3 months-4
years) were evaluated for MR. Although they presented multiple dysmorphic
features and two of them had congenital anomalies (heartrenal),
they could not be connected to any known genetic syndrome
in all cases. Karyotype by G-banding was normal. HR-CGH was then
performed in DNA samples labelled with FITC dUTPs (nick translation).
Normal DNA, labelled with Texas Red, was used as reference
DNA. The results were analysed with the Applied Imaging software.
Results: Four of the five patients had redundant chromosomal material:
1) 46,XX, ish enh (1)(p31p34), enh(13)(q22), 2) 46,XX, ish enh
(12p13.3), 3) 46,XX, ish cgh enh (1)(q24) and 4) 46,XX, rev ish enh
(16p12p13.1) and 5) the fifth patient had a subtelomeric deletion [ish
cgh 46,XX, del (18)(q21.1qter)]. For the confirmation of the results, the
patients’ parents were analysed with the same technique and were
found normal.
Conclusions: HR-CGH contributes to the detection of chromosomal
aberrations along with conventional karyotype, FISH analyses and
quantative molecular methods are a useful adjunct and are well suited
in the diagnosis of mentally retarded and dysmorphic patients.
P0343. Stage-by-stage study of genome-wide DNA methylation
patterns in metaphase chromosomes of human preimplantation
embryos
A. A. Pendina 1 , O. A. Efimova 2 , O. A. Leont’eva 1 , I. D. Fedorova 1,2 , T. V.
Kuznetsova 1 , V. S. Baranov 1 ;
1 D.O.Ott’s Institute of Obstetrics & Gynecololgy RAMS, Saint-Petersburg, Russian
Federation, 2 Saint-Petersburg State University, Saint-Petersburg, Russian
Federation.
We aimed to study DNA methylation patterns of metaphase chromosomes
in human preimplantation embryos, which allows establishment
of differences between parental genomes and DNA remethylation timing
for individual chromosome loci. 83 metaphases from 54 IVF triploid
and abnormal diploid human embryos from zygote to blastocyst
stage have been analyzed by immunofluorescence with monoclonal
anti-5-methylcytosine antibodies(Eurogentec). At the first cleavage
one homologue was more or less methylated than another one(s), indicating
different degree of methylation of parental genomes. In twocell
embryos all chromosomes were hemimethylated - one chromatid
was more methylated than another one. Homologues differed only in
methylation degree of old chromatids. Newly synthesized chromatids
were hypomethylated in all homologues, showing that differential
DNA methylation of parental genomes is not maintained any more.
Hypomethylation affected all regions along chromosome arms with
no band-specificity. Hemimethylated chromosomes were typical up to
blastocyst stage, but their number decreased during subsequent divisions.
Chromosomes with both hypomethylated chromatids appeared
at four-cell stage and increased in number up to morula stage. Surprisingly,
since four-cell stage proportion of hemimethylated and hypomethylated
chromosomes varied from blastomere to blastomere, with
no preference of certain chromosome being hemi- or hypomethylated.